Vibrio parahaemolyticus, a Gram-negative bacterium, is a natural inhabitant of estuarine and coastal environments. In humans, this pathogen is a globally important cause of acute gastroenteritis. It's a multi-serotype pathogenic bacteria, and can be classified into 13 O serotypes and 71 K serotypes based on somatic (O) antigens and capsular (K) antigens (Han et al., 2008). Since 1997, the pandemic isolates, including serotype O3:K6 and its serovariants, have spread globally by either sporadic diarrhea or contaminated food-related outbreaks (Nair et al., 2007; Chowdhury et al., 2013).

All the pandemic isolates share the following specific genetic markers: the presence of the thermostable direct hemolysin(tdh) gene, the absence of the TDH-related hemolysin(trh) gene, and a distinctive toxRS sequence (toxRS/new), which can be amplified by a group-specific PCR (GS-PCR) (Matsumoto et al., 2000). Until 2007, 22 serotypes had been traced to the pandemic clone based on these characteristics (Nair et al., 2007). In recent years, an increasing number of pandemic serotypes have been reported to be widely distributed in countries across four continents (Asia, Europe, the Americas and Africa) (Ansaruzzaman et al., 2008; Ottaviani et al., 2010; Powell et al., 2013; Li et al., 2014; Velazquez-Roman et al., 2014; Guerrero et al., 2017), suggesting that these pandemic serotypes pose a mounting public health threat. This threat calls for higher surveillance of the pandemic clone to reduce illnesses.

In China, V. parahaemolyticus has been the leading cause of foodborne outbreaks and bacterial infectious diarrhea since the 1990s, especially in coastal regions (Gao et al., 2016; Li et al., 2016). The pandemic serotype O3:K6 was first documented as the dominant serotype in 2002 and was proven to be a pandemic clone in 2008 (Vongxay et al., 2008). During the period of 2007–2012, approximately 56% of the clinical isolates in southern coastal areas of China had pandemic characteristics (Li et al., 2014). In one of our multi-center active surveillance programs, we found that 63.3% of the tested isolates were pandemic isolates in southeastern China from 2009 to 2013 (Chen et al., 2016).

The above findings indicate that the pandemic clone of V. parahaemolyticus plays an important role in causing infectious diseases in China. However, although laboratory-based surveillance for acute infectious diarrhea has been established in several coastal regions (Yu et al., 2011; Zhang et al., 2014; Li et al., 2015), a nationwide study or review of the distribution of infections caused by the pandemic isolates of V. parahaemolyticus has not been conducted in this country. Thus, a full understanding of the spread of this unique clone is needed to prevent outbreaks and sporadic illnesses in China.

In this study, we identified Chinese pandemic isolates of V. parahaemolyticus mostly from published literatures, and re-analyzed the sero-prevalence and genetic diversity of these pandemic isolates as a whole. We isolated some of these pandemic isolates in our active studies of diarrheal infection, and mainly focused on their antimicrobial responses in this study, which has not been shown previously. Overall, our intention is to generate a comprehensive overview of the spread of pandemic Vibrio parahaemolyticus O3:K6 and its serovariants in China since the emergence of this clone.

As an emerging public health concern, pandemic V. parahaemolyticus infection has attracted wide attention from scientists (Jun et al., 2014; Velazquez-Roman et al., 2014; de Jesús Hernández-Díaz et al., 2015). The present study has provided an overview of the prevalence of pandemic isolates of V. parahaemolyticus in both clinical and environmental samples collected from multiple Chinese studies. We demonstrated that these pandemic isolates showed high serotypic and genetic diversity. The O3:K6 pandemic isolates (persistent for 11 years from 2002 to 2012) spread across 11 provinces indicate that the pandemic clone has been endemically established in China. Continued monitoring of antibiotic resistance patterns in pandemic isolates is urgently needed to avoid the excessive misuse of antibiotics, although most of the isolates tested in this study only showed high resistance to ampicillin.

The serovariants of this pandemic clone were abundant and variable. From 1996 to 2007, up to 22 pandemic serovariants were identified around the world (Nair et al., 2007), suggesting that the pandemic isolates could easily adapt new serotypes to survive in new environments. In this study, we found that 27 pandemic clinical serotypes and four environmental serotypes have been identified in the collected Chinese isolates. This is a significant number when compared to the number of serotypes found in other countries or regions (Nair et al., 2007; Pazhani et al., 2014; Velazquez-Roman et al., 2014), although the regional persistence of O3:K6 pandemic isolates has also been discovered in many other countries, such as Peru (Gil et al., 2007), Chile (Ansede-Bermejo et al., 2010), Japan (Okuda et al., 1997), India (Pazhani et al., 2014), and Thailand (Mala et al., 2016). To the best of our knowledge, no study has explained why serotypes of the pandemic clone are so diverse in China. Researchers around the world have made some progress in finding the cause of such serodiversity of the pandemic clone. Such research has determined that the O- and K-antigens are mostly mutated concurrently by horizontal gene transfer (HGT) (Okura et al., 2008), and it is speculated that new serovariants have emerged from the pandemic O3:K6 strains via replacement of the putative O and K antigen gene clusters (Okura et al., 2008; Harth et al., 2009; Chen et al., 2010). This is important for the survival of the pandemic clone in the face of changing external environments and host immunological resistance.

The analyzed isolates in this study were distributed in regions where the differences of temperature and other environmental factors are vast. Therefore, we suspect that the strains must adapt to different living environments in the transmission process by altering their biological properties more frequently. Making serological changes may be one of the most efficient ways for this to be accomplished. However, the specific mechanism of serotype conversion is not yet known. The highest priority is currently to continuously monitor the emergence of new serovariants to prevent the pandemic strains from causing outbreaks along the coastline and spreading to other countries and regions.

MLST is known to provide greater resolution than stereotyping. In a previous study, we provided an extensive review of isolates found in Chinese patients, and the results indicated a high degree of genetic diversity and a complicated population structure of V. parahaemolyticus (Han et al., 2015). With the present MLST study, we intended to demonstrate genetic similarities or differences among the pandemic isolates identified from different sources. As expected, we found that most of the pandemic STs were classified into the same clonal complex (CC3). However, some molecular divergence was also found. All the pandemic ST88 in Zhejiang province were assigned to CC345 (not CC3), and ST302, which originated in Shanghai province, was identified as a singleton with no relationship to other pandemic STs. Singletons do not seem to belong to the same clone as other pandemic isolates, but they do share the pandemic traits (toxRS/new+, tdh+, and trh−).

Chen et al. demonstrated the isolates of pandemic ST302 were clustered with other pandemic isolates based on other molecular typing methods, such as enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) (Chen et al., 2012). Similarly, in our pulsed field gel electrophoresis (PFGE) analysis (data not shown), the pandemic isolates of ST88 and ST3 shared the same PFGE profile. Thus, we observed that current typing and clustering methods may lead to controversial results, making it difficult to draw conclusions.

Therefore, a combined application of several molecular typing techniques should be considered in epidemiological investigations of V. parahaemolyticus pandemic isolates. As explained before (in Materials and Methods section), WGS is the best way to accurately portray the evolution and population structure of V. parahaemolyticus isolates at the molecular level (Cui et al., 2015; Haendiges et al., 2015), but the high cost limits its popularity in the analysis of large quantities of specimens.

Another important aspect of this study was the investigation of the antimicrobial susceptibility of different pandemic isolates. Our results revealed similar antibiotic susceptibility profiles in pandemic ST3, ST88, and non-pandemic isolates. This finding was similar to that in the work of Elmahdi et al. (2016). They concluded that the sampling location or month in which the samples were collected did not significantly impact V. parahaemolyticus resistance patterns because isolates from both environmental and clinical sources shared similar antibiotic resistance profiles. Unsurprisingly, the majority of the isolates tested in this study showed ampicillin resistance, which is very common in V. parahaemolyticus isolates recovered from different sources (Sun et al., 2013; de Jesús Hernández-Díaz et al., 2015; Elmahdi et al., 2016; Mala et al., 2016). This result suggests that ampicillin should have a negligible role in the treatment of V. parahaemolyticus infection. In fact, a survey conducted in the United States showed that very high V. parahaemolyticus ampicillin resistance could be traced as far back as 1978 (Blake et al., 1979).

In contrast, most of the isolates tested were sensitive to the majority of antibiotics tested, and all isolates were susceptible to LVX, MEM, IPM, FEP, and CAZ. This result suggests that these drugs can be used as an alternative antibiotic therapy. It must be noted that recently isolated V. parahaemolyticus strains, including pandemic strains, have displayed resistance to multiple antibiotics (Jun et al., 2012, 2014), which increases concerns about possible antibiotic treatment failure. Although we did not discover any multidrug resistant bacteria (MDR) isolates in this study, continued monitoring of pandemic strain susceptibility to antibiotic resistance is urgently needed to avoid the excessive misuse of antibiotics used to treat infections that pose threats to public health.

Our findings represent a comprehensive review of the pandemic V. parahaemolyticus O3:K6 and its serovariants by thoroughly assessing an extensive collection of clinical and environmental pandemic isolates from multiple Chinese studies. High levels of serotypic and genetic diversity in the pandemic clone are found, which suggests that the involved regions are becoming important reservoirs for the emergence of novel pandemic strains, which makes the clinical management of the infection and its prevention potentially challenging. Thus, we underscore the need for routine clinical and environmental monitoring to prevent pandemic V. parahaemolyticus infection and dissemination, including monitoring of antimicrobial response even though most current antimicrobial agents in routine use are effective. The mechanism in which the isolates undergo seroconversion with pandemic genetic marks warrants extended investigation.

Conceived and designed the experiments: DH and CH. Performed the experiments: DH, FY, and HT. Analyzed the data: DH, CW, and PZ. Contributed reagents/materials/analysis tools: DH and CR. Wrote the paper: DH.